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Abstract:

Disclosed herein is a composition and method for sample preparation of
proteins for their size separation by electrophoresis, suitable for
molecular-weight determination of proteins in the range between about
14,000 and 500,000. In an embodiment, proteins, particularly those
exhibiting biased migration, are modified to change their intrinsic
charge, or carbohydrate component to improve accuracy of their molecular
weights as determined by electrophoretic size separation via their
interaction with ionic surfactants. In a preferred embodiment, the
proteins are carbamylated with potassium cyanate and their carbohydrate
components are oxidized with sodium periodate.

7. A protein denaturing composition of claim 1, wherein said
dithiodialkylcarboxylic acid is selected from the group of dithioacids
consisting of dithiodiglycolic acid, dithiodipropionic acid, and
dithiodibenzoic acid.

8. A protein denaturing composition of claim 1, wherein said
low-fluorescence isothiocyanate is selected from the group of compounds
consisting of sulfophenyl isothiocyanate, carboxyphenyl isothiocyanate,
disulfophenyl isothiocyanate, trisulfophenyl isothiocyanate, and
dicarboxyphenyl isothiocyanate.

15. A method for sample preparation of proteins of claim 14, wherein said
mobility modifier is potassium cyanate.

16. A method for sample preparation of proteins of claim 14, wherein said
mobility modifier is sodium periodate.

17. A method for capillary sieving electrophoresis with cationic
surfactant for size separation of proteins, consisting of the steps: a)
Preparing the protein sample by heating the protein solution at
40.degree. C.-100.degree. C. for 1-60 min with about 1%
cetyltrimethylammonium chloride, about 1% tris(carboxyethyl)phosphine,
and protein mobility modifier, said mobility modifier selected from the
group of reagents consisting of i)
1-ethyl-3-.beta.-dimethylaminopropyl)carbodiimide hydrochloride; ii)
acetic anhydride, aconitic anhydride, citraconic anhydride, phtalic
anhydride, succinic anhydride, glutaric anhydride, itaconic anhydride,
propionic anhydride; iii) salt of periodic acid; iv) salt of cyanic acid,
salt of sulfocyanic acid; v) dithiodialkylcarboxylic acid,
dithiodialkylamine; vi) low-fluorescence isothiocyanate; vii)
low-fluorescence succinimidyl ester; viii) low-fluorescence sulfonyl
chloride; ix) low-fluorescence dichlorotriazine; and x) low-fluorescence
tetrafluorophenylester; b) rinsing the separation capillary; c) filling
the capillary with a separation medium for capillary electrophoretic size
separation of proteins, said separation medium consisting of: i) a
cationic surfactant; ii) an acidic buffer; and iii) a sieving polymer,
wherein said sieving polymer is selected from the group consisting of
linear polyacrylamide, poly(dimethyl acrylamide), poly(hydroxyethyl
acrylamide), poly(hydroxypropyl acrylamide), poly(ethoxyethyl
acrylamide), poly(vinyl alcohol), poly(vinyl pyrrolidone), hydroxyethyl
cellulose, scleroglucan, guaran, locust bean gum, glucomannan, pullulan,
dextran, and poly(ethylene oxide), with a proviso that when said sieving
polymer is poly(ethylene oxide), it is in the concentration from about 16
g/L to about 60 g/L; d) sample injection, wherein the capillary inlet is
washed by a triple immersion in distilled water, then the capillary inlet
and cathode are immersed in the sample, capillary outlet and anode are
immersed in a vial containing separation medium, and finally an injection
voltage from about 0.5 kV to about 12 kV is applied between the anode and
cathode for about 1 s to about 60 s; e) separation, wherein the capillary
inlet and cathode are immersed in a vial containing said separation
medium, capillary outlet and anode are immersed in other vial containing
said separation medium, then a separation voltage from about 1 kV to
about 20 kV being applied between the anode and cathode for about 1
minute to about 20 minutes; and f) detection, wherein absorption of
monochromatic light having wavelength from about 210 nm to about 420 nm
is measured and plotted in electropherogram for further data analysis.

18. A method for capillary sieving electrophoresis with cationic
surfactant of claim 17, wherein said mobility modifier is about 100 mM
potassium cyanate.

19. A method for capillary sieving electrophoresis with cationic
surfactant of claim 17, wherein said mobility modifier is about 100 mM
sodium periodate.

[0054] The present invention relates to electrophoretic size separation of
proteins in sieving media, wherein one or more ionic surfactants form
charged complexes with the proteins, equalize their surface charge
density, make them migrating in sieving media independently of their
intrinsic charge, and allow their size separation and molecular-weight
determination. Specifically, the invention is directed to the sample
preparation of proteins anomalously migrating in the presence of ionic
surfactants, which modifies those anomalously migrating proteins and
normalizes their electrophoretic migration.

BACKGROUND OF THE INVENTION

Electrophoresis in Sieving Media

[0055] Electrophoretic sieving media are used to size separate
biopolymers: nucleic acids, polysaccharides, and proteins. They provide a
system of obstacles (typically gel or entangled polymers) in the
electrophoretic migration path so that the migrating biopolymers collide
with the obstacles and these collisions suppress their apparent migration
velocity. (The first electrophoretic sieving media were starch and
polyacrylamide gel.) The size separation is based on the fact that the
electrophoretic migration of larger molecules and particles is retarded
more than that of small molecules. Nucleic acids are equally ionized at
non-acidic pH and have sufficient charge and mobility. They need not be
modified to size separate during electrophoretic migration in sieving
media. On the other hand, protein ionization and charge significantly
vary depending on the amino acid composition. Therefore, native proteins
are not size separated in sieving media in the absence of ionic
surfactants. However, when heated with an ionic surfactant, proteins
denature and bind the ionic surfactant, generating complexes with more or
less equal surface charge density. These complexes migrate in sieving
media according to their size.

Slab Gel Electrophoresis

[0056] SDS electrophoresis in polyacrylamide slab gel (SDS PAGE) was the
first method separating proteins according to their size1-4. Shortly
after the invention of SDS PAGE, a method separating proteins by
polyacrylamide gel electrophoresis (PAGE) in the presence of cationic
surfactants was described5. A study observing the migration behavior
of protein-cationic-surfactant-complexes followed, predicting a failure
of the electrophoresis in the presence of cationic surfactants to
determine molecular weights of proteins6. Later, cetylpyridinium
chloride7 and cetyltrimethylammonium bromide8-12 were used for
size separations of proteins by PAGE. Several protocols have been
developed to denature proteins with cetyltrimethylammonium
bromide8-12.

Capillary Electrophoresis

[0057] When electrophoresis of proteins in sieving media was transferred
from slab gels into capillaries, crosslinked polyacrylamide gel was
initially used as a sieving matrix13,14. When linear hydrophilic
polymers were introduced as a replaceable sieving matrix for separation
of polynucleotides15, various polymers were utilized as a sieving
matrix for electrophoretic size separation of biopolymers: linear
polyacrylamide16-18, poly(ethylene oxide)19, dextran16,
guaran20, glucomannan21, poly(vinyl alcohol)22,
poly(hydroxypropyl acrylamide)23, poly(ethoxyethyl
acrylamide)24, agarose25, and pullulan26. Size separations
of proteins by capillary electrophoresis were performed mostly by SDS
capillary sieving electrophoresis (CSE) in the molecular-weight range
between about 14,000 and 205,000. The method was also modified for the
size separation of proteins on microchip27 with poly(dimethyl
acrylamide) as a sieving polymer28. Capillary electrophoresis meant
a number of advantages as compared to electrophoresis in slab gel: faster
analysis, automation, higher separation efficiency, and higher detection
sensitivity. Nevertheless, a small size of capillaries emphasized the
effect of the capillary wall: typically fused silica capillaries were
used that contained ionized silanol groups on their internal surface,
resulting in strong wall adsorption, significant electroosmotic flow,
eddy migration, and consequent mediocre separation efficiency.
Electroosmotic flow was eventually suppressed by applying a
hydrolytically stable neutral coating on the capillary wall (U.S. Pat.
No. 5,143,753). Nevertheless, in SDS CSE, SDS adsorbs on the neutral
coating and generates secondary electroosmotic flow. Mediocre
reproducibility and separation efficiency are the results of this
deleterious effect. Currently, SDS CSE is performed in bare capillaries
after extensive rinsing of the capillary between runs, significantly
reducing the throughput of the analysis (U.S. Patent Application
20090314638). Hypothetically, electroosmotic flow in SDS CSE could be
also suppressed by reducing pH of the sieving medium and a consequent
suppression of the silanol ionization in the capillary wall. However, SDS
binding of proteins is weaker at pH<6 and SDS electrophoresis at this
pH results in significantly broader peaks29 excluding this
alternative from a real world practice.

Anomalous Protein Migration

[0058] Anomalous migration of some proteins was observed already in the
early years of SDS PAGE when lysozyme and ribonuclease A did not migrate
as expected from their molecular weights1,4,5. The authors
speculated the anomalous proteins did not completely unfold and/or were
not saturated with SDS4. Later it was found that even a single
substitution of a neutral amino acid in α-crystallins resulted in
changed mobility in SDS and thus different molecular weight30.
Electrostatic repulsion between SDS and strongly acidic proteins could
have been the cause for lower SDS binding31. Also glycoproteins were
proposed to bind SDS below its saturation since hydrophilic carbohydrates
were not likely to strongly bind SDS. William and Gratzer hypothesized
the anomalously slow migration of acidic ferredoxins in SDS PAGE was
caused by insufficient surfactant binding due to electrostatic repulsion
of SDS and protein carboxylic groups5. This idea was corroborated by
an observation that some acidic proteins, such as pepsin, papain, and
glucose oxidase did not bind measurable amount of SDS32. Similarly,
maleylation of cyanogen bromide fragments of cytochrome c significantly
reduced their apparent molecular weights while that of native cytochrome
c was not significantly affected by carbamylation33. Lysozyme was
also modified by a reaction with dithio-compounds with various
charges4. Carboxyethyl-, hydroxyethyl-, and aminoethyl-lysozyme
derivatives migrated more anodically than lysozyme itself in 8 M urea, in
absence of any ionic surfactant. The mobility differences in SDS PAGE
indicated the intrinsic charge had an effect on the amount of SDS bound
to the proteins.

[0059] Guttman and Nolan investigated the accuracy of molecular weights of
65 proteins as determineded by SDS electrophoresis in capillary and slab
gel format. Independently of the format, more than one fourth of proteins
exhibited biased migration34.

Normalization of Biased Molecular Weights

[0060] It was suggested to use so called Ferguson plot to correct the
molecular weight of proteins with biased migration35,36.

[0061] Based on the hypothesis that electrostatic repulsion between ionic
surfactant and proteins cause biased migration in electrophoretic size
separation, several methods have been tested to normalize protein
migration. The anomalously slow migration of acidic ferredoxins in SDS
PAGE was normalized by esterification of their carboxyl groups with
methanols.

[0062] Deglycosylation of several glycoproteins with N-glycosidase F
improved the accuracy of molecular weights of these glycoproteins in SDS
PAGE and SDS CSE on microchip37.

[0063] Several reaction schemes have been used to modify proteins to
detect them by laser fluorescence detection. Some of them can be used for
sample preparation to normalize protein migration in electrophoresis in
the presence of a ionic surfactant.

Modifying Proteins by Carbamylation of Amino Groups

[0064] Protein amino groups can be modified by carbamylation with cyanate
when homocitrulline is formed38

[0065] Proteins can be modified on their carboxylic groups by a reaction
with a water-soluble carbodiimide, e.g.,
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC,
(CH3)2--N--(CH2)3--N═C═N--CH2--CH3)-
. Unstable acylurea ester is formed first then it reacts with a primary
amine38

[0066] The participation of a primary amine in the reaction also means the
reaction can be hypothetically used to modify protein amino groups by a
reaction with a carboxylic acid and EDC.

Modifying Proteins on Amino Groups by Reaction with Isothiocyanate
Derivatives

[0067] The reaction of protein amino groups with isothiocyanate
derivatives has been widely used to label proteins with a fluorescent dye
for their laser-induced fluorescence detection in HPLC and other
separation methods. Isothiocyanates react with primary amines forming
thiourea derivatives39

R1N═C═S+Protein-NH2→RiNH--CS--NH-Protein

Modifying Proteins on Amino Groups by a Reaction with Succinimidyl Ester
Derivatives

[0068] The reaction of protein amino groups with succinimidyl ester
derivatives has been also used for labeling proteins with a fluorescent
dye. Succinimidyl ester reacts with primary amines and forms carboxamide
derivatives39

Modifying Proteins on Amino Groups by a Reaction with Sulfonyl Chloride
Derivatives

[0069] Another reaction of protein amino groups that is to label proteins
is with sulfonyl chloride derivatives. Sulfonyl chlorides react with
primary amines and form sulfonamide derivatives39

R1--SO2Cl+R2NH2→R1--SO2--NH--R2

Modifying Proteins on Amino Groups by a Reaction with Aldehyde
Derivatives

[0070] The reaction of protein amino groups with aldehyde derivatives has
been also used for labeling proteins with a fluorescent dye when a Schiff
base is formed first and then it is reduced to a corresponding
alkylamine39

R1CO--H+R2NH2→RiCH═N--R2+H2O→-
RiCH2--NH--R2

Modifying Proteins on Sulfhydryl Groups by a Reaction with Charged Dithio
Derivatives

[0071] Proteins can be modified by reaction of their disulfide bridges
with dithioderivatives in the presence of an excess thiol, where protein
thiols are generated

R1SH+R2--S--S-Protein→R1--S--S--R2+HS-Protein

[0072] The protein thiols then react with disulfides generating proteins
modified in their disulfides19

HS-Protein+R3--S--S--R3→R3--S--S-Protein+R3---
SH

[0073] When the modifying disulfide carries a charged moiety the protein
intrinsic charge can be also modified4.

BRIEF SUMMARY OF THE INVENTION

[0074] The present invention is suitable for electrophoretic size
separation of proteins and particularly sample preparation procedure
improving the accuracy of molecular weight determination. Disclosed
herein are the compositions of the reagents modifying proteins and the
method of the protein modifications improving the accuracy of the protein
molecular weights obtained by electrophoretic size separation in the
presence of ionic surfactants.

[0078]FIG. 4 shows the relation between logarithmic molecular weight of
proteins and their electrophoretic mobilities. .diamond-solid.-cytochrome
c, .tangle-solidup.-carbamylated cytochrome c. Experimental conditions
were same as described in FIG. 2.

[0080] We propose to modify proteins during the sample preparation for the
electrophoretic size separation and alter their functional groups and so
suppress the existing charged groups. Moreover, these modifications
reverse the sign of the charge of the charged groups and thus make a
complex formation between proteins and ionic surfactant easier. Similarly
we disclose modification of glycoproteins when the carbohydrate component
is oxidized with periodate.

[0081] We disclose here a protein denaturing composition for sample
preparation of proteins prior their electrophoretic size separation,
consisting of: [0082] a) an ionic surfactant, [0083] b) an electrolyte,
[0084] c) a reducing reagent cleaving disulfidic bridges, [0085] d) a
mobility modifier that reacts with proteins and changes the
electrophoretic mobility of the complex between said protein and said
ionic surfactant.

[0100] Specifically we disclose a protein denaturing composition
consisting of about 0.1 M potassium cyanate, about 1%
hexadecyltrimethylammonium chloride, and about 1%
tris(carboxyethyl)phosphine and also a protein denaturing composition
consisting of about 0.1 M sodium periodate, about 1%
hexadecyltrimethylammonium chloride, about 1%
tris(carboxyethyl)phosphine, and about 100 mM sodium acetate. We further
disclose a protein denaturing composition consisting of about 0.1 M
sodium thioglycolate, about 1% hexadecyltrimethylammonium chloride, and
about 10 mM tris(carboxyethyl)phosphine. We also disclose a protein
denaturing composition consisting of about 0.1 M potassium cyanate, about
1% sodium dodecylsulfate, and about 1% dithiotreitol. We also disclose a
protein denaturing composition consisting of about 0.1 M sodium
periodate, about 1% sodium dodecylsulfate, about 1% dithiotreitol, and
about 100 mM sodium acetate.

[0101] Further we disclose a method for denaturation and sample
preparation of proteins prior to their size separation by
electrophoresis, consisting of the steps: mixing the protein sample with
an ionic surfactant, electrolyte, reducing reagent cleaving disulfidic
bridges, and mobility modifier that reacts with proteins and changes the
electrophoretic mobility of the complex between said protein and said
ionic surfactant; said mobility modifier is selected from the group of
reagents consisting of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
hydrochloride, acetic anhydride, aconitic anhydride, citraconic
anhydride, phtalic anhydride, succinic anhydride, glutaric anhydride,
itaconic anhydride, propionic anhydride, a salt of periodic acid,
particularly sodium periodate, a salt of cyanic acid, particularly
potassium cyanate, a salt of sulfocyanic acid, dithiodialkylcarboxylic
acid, dithiodialkylamine, low-fluorescence isothiocyanate,
low-fluorescence succinimidyl ester, low-fluorescence sulfonyl chloride,
low-fluorescence dichlorotriazine, low-fluorescence
tetrafluorophenylester, and heating the prepared mixture at 40°
C.-100° C. for 1-60 min.

[0102] We also disclose a method for capillary sieving electrophoresis
with cationic surfactant for size separation of proteins, consisting of
the steps: a) Preparing a protein sample by reacting the protein with the
protein mobility modifier, said mobility modifier selected from the group
of reagents consisting of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
hydrochloride, acetic anhydride, aconitic anhydride, citraconic
anhydride, phtalic anhydride, succinic anhydride, glutaric anhydride,
itaconic anhydride, propionic anhydride, salt of periodic acid,
particularly sodium periodate, salt of cyanic acid, particularly
potassium cyanate, salt of sulfocyanic acid, dithiodialkylcarboxylic
acid, dithiodialkylamine, low-fluorescence isothiocyanate,
low-fluorescence succinimidyl ester, low-fluorescence sulfonyl chloride,
low-fluorescence dichlorotriazine, low-fluorescence
tetrafluorophenylester, b) rinsing the separation capillary; c) filling
the capillary with a separation medium for capillary electrophoretic size
separation of proteins, said separation medium consisting essentially of
a cationic surfactant; an acidic buffer; and a sieving polymer, wherein
said sieving polymer is selected from the group consisting of linear
polyacrylamide, poly(dimethyl acrylamide), poly(hydroxyethyl acrylamide),
poly(hydroxypropyl acrylamide), poly(ethoxyethyl acrylamide), poly(vinyl
alcohol), poly(vinyl pyrrolidone), hydroxyethyl cellulose, scleroglucan,
guaran, locust bean gum, glucomannan, pullulan, dextran, and
poly(ethylene oxide), with a proviso that when said sieving polymer is
poly(ethylene oxide), it is in the concentration from about 16 g/L to
about 60 g/L; d) Sample injection, wherein the capillary inlet is washed
by a triple immersion in distilled water, then the capillary inlet and
cathode are immersed in the sample, capillary outlet and anode are
immersed in a vial containing separation medium, and an injection voltage
from about 0.5 kV to about 12 kV is applied between the anode and cathode
for about 1 s to about 60 s; e) separation, wherein the capillary inlet
and cathode are immersed in a vial containing said separation medium,
capillary outlet and anode are immersed in other vial containing said
separation medium, then a separation voltage from about 1 kV to about 20
kV being applied between the anode and cathode for about 1 minute to
about 20 minutes; f) detection, wherein absorption of monochromatic light
having wavelength from about 210 nm to about 420 nm is measured and
plotted in electropherogram for further data analysis.

EXAMPLES

[0103] The separations described in these examples were performed in 3D CE
capillary electrophoresis instrument at 20° C. in a bare or coated
capillary of internal diameter 75 μm and outer diameter 360 μm with
UV detection at 214 nm or 420 nm

Example 1

[0104] Carbamylation of cytochrome c for an improved accuracy of molecular
weights by capillary sieving electrophoresis with cationic surfactant

[0105] 5 mg bovine heart cytochrome c was dissolved in 820 μL water and
mixed with 40 μL of 25% cetyltrimethylammonium chloride, 40 μL of
10% tricarboxyethyl phosphine, and 100 μL of 1 M KCNO. After
dissolving, the mixture was heated at 70° C. for 20 min and then
cooled to room temperature.

Example 2

[0106] Oxidation of ovalbumin with NaIO4 for an improved accuracy of
molecular weights by capillary sieving electrophoresis with cationic
surfactant

[0107] 5 mg of ovalbumin was dissolved in 1 mL of 0.1 M sodium acetate
buffer, pH 5.5 and mixed with 75 μL of 0.2 M NaIO4 and 50 μL
of 25% cetyltrimethylammonium chloride. The mixture was heated at
40° C. for 10 min. Then 50 μL of 50% glycerol was added.

Molecular Weight of Proteins Determined by Capillary Sieving
Electrophoresis with a Cationic Surfactant

[0110] Protein mobilities μ were calculated from equation (1)

μ = L t × L eff V × t m ( 1 )
##EQU00001##

where Lt is the total capillary length, Leff is the effective
capillary length, V is the applied voltage and tm is the migration
time of protein. Logarithmic molecular weights of proteins were plotted
against the calculated mobilities. Using suitable proteins as molecular
weight standards, a calibration curve was plotted (FIG. 4) and an
equation, which describes the plot, was used to calculate the apparent
molecular weights of the tested proteins. Cytochrome c with a true
molecular weight 12,400 exhibited apparent molecular weight of 56,200,
i.e., the error of the molecular weight was about 350% whereas the
carbamylated cytochrome c, having approximately the same true molecular
weight, showed the apparent molecular weight of 12,100, which corresponds
to a relative error of -2.3%.

Example 6

Ferguson Plot for Proteins Separated by Capillary Sieving Electrophoresis
with a Cationic Surfactant

[0111] To measure the dependence of protein mobilities on the
concentration of the sieving polymer in capillary sieving electrophoresis
with a cationic surfactant, the electrophoretic separation was performed
in 0-16 g/L polyethylene oxide (Mw, 600 k), 100 mM β-alanine
glutamate, 0.2% cetyltrimethylammonium chloride. A bare capillary, 75
μm ID, 360 μm OD, total length 335 mm, effective length 250 mm, was
flushed with distilled water and filled with a composition containing
0-16 g/L polyethylene oxide (Mw, 600 k), 100 mM β-alanine
glutamate, 0.2% cetyltrimethylammonium chloride. Proteins were injected
electrokinetically at +8 kV for 15 s, separated at +10 kV for 20 min, and
detected by UV adsorption at 214 nm. The protein mobilities μ were
calculated from the equation 1 and plotted in the Ferguson plot as a
function of the sieving polymer concentration (FIG. 5). The Ferguson plot
showed the anomalous migration of native cytochrome c and the proper
migration of carbamylated cytochrome c.